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Title: Cell wall synthesis in Bacillus subtilis
Author: Sassine, Jad
ISNI:       0000 0004 7429 9121
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2017
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The peptidoglycan (PG) layer is responsible for maintaining cell shape and permitting cell division in almost all bacteria. Made of glycan chains connected by short peptides, PG forms a net-like structure surrounding the cytoplasmic membrane. Membrane-anchored PG synthases, called penicillin-binding proteins (PBPs), synthesize PG during cell growth and division by utilising the precursor lipid II but the molecular mechanism of these processes in Bacillus subtilis are largely unknown. The genetic and phenotypic analysis of B. subtilis has shown that PG synthesis and cell division are modulated by components of the central carbon metabolism (Weart et al., 2007). In particular, UgtP, which synthesises the glucolipid precursor for the lipoteichoic acid, has been suggested to function as a metabolic sensor governing cell size. However, the mechanism by which UgtP impacts cell wall synthesis remained unknown. Here we have constructed different B. subtilis strains with deletions in cell wall synthesis and/or carbon metabolism genes. Cells lacking the LTA precursor glucolipid grew with similar rate as wild type cells but were shorter and wider. The overexpression of ugtP caused filamentation, supporting the hypothesis that UgtP inhibits FtsZ polymerization (Weart et al., 2007). The ugtP mutant had increased level of several PG precursors and mild alterations in PG composition suggesting an increased DL-endopeptidase activity. Combining ugtP deletion/depletion with deletions with several cell wall genes resulted in morphological effects. The deletion of the PBP1 gene and simultaneous depletion of ugtP resulted in thin and bent cells. The double deletion of ugtP and lytE, a hydrolase important for cell elongation, produced shorter bent cells with severe shape defects. These results suggest that the function of UgtP contributes to balanced cell wall synthesis and hydrolysis. We also characterised several crucial cell wall enzymes. The depletion of the essential PBP2B caused cell division defects followed by lysis. Interestingly, cells expressing a catalytically inactive PBP2B were viable, but they required functional PBP3, a homologue of PBP2B that is dispensable in wild-type cells. PBP3 showed enhanced septal localisation in a strain with inactive PBP2B, but this strain produced aberrant septa. Biochemical assays were used to characterize for the first time the activities and interactions of PBP1, PBP2B, and PBP3. Novel interactions between these PBPs and with the lytic transglycosylase homologue YrrL were detected. In summary, this work contributes to our understanding of the PG synthesis during cell division.
Supervisor: Not available Sponsor: Marie Skłodowska-Curie Actions ; European Commission
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available